The Signal Transducer and Activator of Transcription 3 (STAT3) is persistently activated in many types of cancers, including breast, prostate, leukemia and lymphoma, to support oncogenesis. Suppression of STAT3 by RNAi not only leads to apoptosis of tumor cells but also activation of the immune system in the tumor microenvironment to promote tumor cell eradication. These results strongly suggest that inhibiting STAT3 activity is a potent and broadly applicable cancer therapeutic strategy. In addition to high throughput screening programs for the discovery of STAT3 inhibitors, major efforts have focused on peptidomimetics based on the phosphopeptides derived from the gp130 subunit of the IL-6 receptor or phosphorylated STAT3, but no drug has reached the clinic yet. A major deficiency in these efforts is the complete absence of atomic resolution structure of STAT3 in complex with any of the inhibitors developed to date. Therefore, rational development and improvement of STAT3 inhibitors has been hampered. In preliminary studies, we have established the conditions to obtain high quality nuclear magnet resonance (NMR) spectra (methyl-13C-1H TROSY) on the 68 kDa monomeric core of a STAT3 construct that encompasses both the SH2 and DNA-binding domains. This is a major breakthrough that allows the investigation of atomic resolution structural information of STAT3-inhibitor complexes using state-of-the-art NMR methods. Recent developments in solution NMR methods have enabled the investigation of both structure and dynamic properties of proteins and their complexes that are of the sizes of STAT3 and even larger. We have also used fragment-based NMR screening methods to identify novel lead small molecule inhibitors of STAT3. These compounds inhibit proliferation of breast and prostate cancer cell lines that contain persistently activated STAT3. We propose to conduct structural studies on STAT3-ligand interactions and further develop the small molecule inhibitors a family of new cancer therapeutic leads. The proposed study will not only develop a novel NMR approach that is applicable to other large proteins but will also provide NMR resonance assignments of STAT3 that will enable structural studies of STAT3-ligand interactions in solution to advance cancer therapeutics development targeting STAT3.